Project 1 builds upon a key observation made in SIDS brainstems in the last grant cycle that serotonergic (5-HT) receptor binding is abnormal in regions of the medulla that contain 5-HT neurons and that are thought to be critical in the modulation of state-dependent, homeostatic reflexes. For the next cycle, we propose that these 5-HT receptor binding abnormalities are not necessarily the primary and/or only defect in 5-HT neurotransmission in these cases. Rather, they may represent a marker of primary and/or other defects elsewhere in 5-HT neurons, e.g., in the expression of the serotonin transporter (SERT), and/or in the neuropeptides substance P (SP) and/or thyrotrophin releasing hormone (TRH) that are known to colocalize with 5-HT and are its key neuromodulators. The proposed studies are novel in that they involve an indepth characterization of the neurochemical organization of the medullary 5-HT system in infancy, the peak age range of SIDS, and of its pathology in SIDS infants. We are especially interested in nicotinic receptor-related neuroanatomy in the medullary- 5-HT system in human gestation due to epidemiologic studies that link maternal cigarette smoking during pregnancy and increased risk for SIDS, and our finding of an association between maternal cigarette smoking during pregnancy and lowered 5-HT receptor binding in the arcuate nucleus, a ventral surface component of the medullary 5-HT system. In Specific Aim 1, we will determine the profile of SERT expression in the human brainstem across early development. In Specific Aim 2, we will determine the brainstem expression of SERT in SIDS cases compared to controls. In Specific Aim 3, we will determine the neurochemical organization of the medullary 5-HT system in the human infant relative to the inter-relationships of 5-HT neurons with SP and TRH. In Specific Aim 4, we will determine the developmental profile of receptor binding to SP and TRH in the medullary 5-HT system in SIDS cases compared to age-matched controls. In Specific Aim 5, we will determine the neuroanatomic inter-relationships between nicotinic receptors and 5-HT neurons in the human medulla during gestation. These studies will utilize tissue receptor autoradiography, single- and double-labeling immunocymchemistry, and in situ hybridization to mRNA transcripts with 2- and 3-dimensional, computer-based graphics and quantitation in alternate tissue sections from the same cases. Further characterization of abnormalities in the medullary 5-HT system in SIDS cases should help lead to the development of strategies to clinically identify and prevent them in affected infants, the ultimate goal of this research.